EA043843B1 - ANTI-C5A ANTIBODIES AND THEIR APPLICATIONS - Google Patents

Description

Cross reference to related applications

This application claims priority to U.S. Provisional Patent Application No. 62/475573, filed March 23, 2017, the contents of which are incorporated herein by reference in their entirety.

Application for government financial support for research or development

The present invention was made with government support under NIH grant AI44970 from the National Institutes of Health (NIH). The government has certain rights to an invention.

State of the art

The complement system is part of the innate immune system, which plays a key role in host defense. However, activated complement also has the potential to cause significant tissue damage and destruction, and dysregulation of complement activity has been found to be associated with a number of rare and common diseases, such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome, rheumatoid arthritis, age-related macular degeneration, etc. Thus, anticomplement therapy is a promising treatment option for these human disorders.

The C5 component of the complement system is a critical protein in the terminal pathway of complement activation and is a precursor protein in the generation of the potent proinflammatory mediator C5a as well as the cytolytic membrane attack complex (MAC), C5b-9.

In some complement-mediated diseases, processes mediated by both C5a and MAC may contribute to pathogenesis, while in other diseases only C5a-mediated inflammation or MAC-mediated cell damage may be involved. Since complement mediators, including C5a and MAC, also play an important role in host defense against pathogenic infection, it is desirable that selective anticomplement drugs, i.e., be developed in the development of therapeutic agents. drugs that block only the negative effects of complement in tissue damage, leaving its normal protective function for the host intact.

The cause of hemolytic disease PNH is MAC. There are other anti-C5 mAbs for the treatment of PNH. However, these antibodies excessively block C5a production, putting patients at greater risk of infection than a therapeutic that blocks only the MAC. Similarly, there are complement-mediated diseases that may be primarily mediated by C5a-dependent inflammation (eg, sepsis), and for such disease conditions, an anti-C5 mAb drug, although expected to be effective, will unnecessarily block MAC in as a side effect.

Thus, there is a need in the art for anti-human C5a mAbs that can inhibit C5a-mediated activity but do not block MAC activity. The present invention addresses these and other needs.

The essence of the invention

In one embodiment, the present invention provides an antibody that specifically binds to C5a. In one embodiment, C5a is human C5a. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a humanized antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an antibody fragment that includes, but is not limited to, Fab, Fab', F(ab) ₂ , F(ab') ₂ and scFv. In some embodiments, the antibody is part of a construct, such as a fusion construct, comprising an antibody and a targeting molecule or effector molecule. In some embodiments, the antibody is part of a conjugate, such as an antibody-drug conjugate.

In one embodiment, the antibody is a chimeric antibody. In one embodiment, the antibody contains at least one of the CDRs selected from the group consisting of

V H-CDR1: SEQ ID NO: 3;

V H-CDR2: SEQ ID NO: 4;

V H-CDR3: SEQ ID NO: 5;

V L-CDR1: SEQ ID NO: 8;

V L-CDR2: SEQ ID NO: 9; And

VL-CDR3: SEQ ID NO: 10, or a variant or variants thereof.

In one embodiment, the antibody comprises a CDR:

V H-CDR1: SEQ ID NO: 3;

V H-CDR2: SEQ ID NO: 4;

V H-CDR3: SEQ ID NO: 5;

V L-CDR1: SEQ ID NO: 8;

V L-CDR2: SEQ ID NO: 9; And

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VL-CDR3: SEQ ID NO: 10, or a variant or variants thereof.

In one embodiment, the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof. In one embodiment, the antibody contains a light chain containing the amino acid sequence of SEQ ID NO: 7 or a variant thereof. In one embodiment, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof, and a light chain containing the amino acid sequence of SEQ ID NO: 7 or a variant thereof. In one embodiment, the antibody is 7A12.

In one embodiment, the present invention provides a method of treating a complement-mediated disease or disorder in an individual, comprising the step of administering to said individual an anti-C5a antibody described herein. In one embodiment, the disease or disorder is at least selected from the group consisting of macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, postoperative syndrome systemic inflammatory response, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria syndrome (PNH), autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, neuromyelitis optica, NMO, multiple sclerosis, delayed graft function , antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS), central retinal vein occlusion (CRVO), central retinal artery occlusion (CRAO), epidermolysis bullosa, sepsis, septic shock, organ transplantation, inflammation (including but not limited to inflammation associated with cardiopulmonary bypass and renal dialysis), C3 glomerulopathy, membranous nephropathy, IgAnephropathy, glomerulonephritis (including, but not limited to, antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, lupus nephritis and combinations thereof), ANCA-associated vasculitis, Shiga toxin HUS, and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), or any combination thereof. In some embodiments, the AR-mediated disease is sepsis, septic shock, rheumatoid arthritis, autoimmune hemolytic anemia, GvHD, antiphospholipid syndrome, or Gaucher disease. In one embodiment, administration of an anti-C5a antibody inhibits the activity of the C5a protein.

In one embodiment, the present invention relates to a method of reducing the activity of an individual's complement system, wherein the method includes administering an antibody to an individual by an administration route selected from the group consisting of enteral administration, parenteral administration, and a combination thereof, and wherein the antibody comprises six complementarity determining regions having the following amino acid sequences: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or a variant or variants thereof. In one embodiment, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, and combinations thereof.

In one embodiment, the present invention describes an anti-human C5a antibody, wherein the antibody has a heavy chain variable region (VH) that has an amino acid sequence that is greater than about 90% (e.g., greater than 91, 92, 93, 94, 95 , 96, 97, 98, 99 or 100%) is identical to SEQ ID NO: 2 or a variant thereof. In one embodiment, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab)2, F(ab')2, scFv, and combinations thereof.

In one embodiment, the present invention describes an anti-human C5a antibody, wherein the antibody has a light chain variable region (VL) that has an amino acid sequence that is greater than about 90% (e.g., greater than 91, 92, 93, 94, 95 , 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 7 or a variant thereof. In one embodiment, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, and combinations thereof.

In one embodiment, the present invention is an anti-human C5a antibody, wherein the antibody has a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region has an amino acid sequence that is more than about 90% (e.g., more is more than about 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 2 or a variant thereof, and wherein the VL region has an amino acid sequence that is more than about 90% identical (e.g. , more than 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 7 or a variant thereof. In one embodiment, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab)2, F(ab')2, scFv, and combinations thereof.

In one embodiment, the present invention provides a cell containing at least one of the antibodies described elsewhere herein. In one embodiment, the cell produces at least one of the antibodies described elsewhere herein. In one embodiment, the cell is a hybridoma.

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In one embodiment, the present invention provides a cell line containing at least one of the antibodies described herein. In one embodiment, the cell line produces at least one of the antibodies described herein. In one embodiment, the cell line is a hybridoma cell line.

Brief description of the figures

The foregoing summary of the invention, as well as the following detailed description of exemplary embodiments of the invention, will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the invention is not limited to the preparation and tools of the embodiments shown in the figures.

In fig. 1 including FIG. 1A and 1B present results showing that mAb 7A12 binds to human C5. In fig. 1A shows the reactivity of 7A12 and 2G1, a control anti-C5 mAb, with intact human C5 assessed using ELISA. The plate was coated with purified human C5. After incubation with serial dilutions of 7A12 or control anti-C5 mAb, bound mAbs were detected using HRP-conjugated rabbit anti-mouse IgG. 7A12 and control anti-C5a mAb showed high reactivity with human C5. In fig. 1B shows, using Western blotting, that mAb 7A12 and control anti-C5a mAb recognized purified human C5 protein under non-reducing and reducing conditions, respectively. The observed 7A12-reactive band of 190 kDa represents the complete C5 protein, while the 115 kDa band represents the C5 chain. NR: non-reducing conditions; R: reducing conditions.

In fig. 2 shows results showing that, unlike the control anti-C5 mAb 2G1, which inhibits red blood cell (RBC) lysis, 7A12 is not active in the hemolysis assay. Antibody-sensitized sheep red blood cells were incubated with normal human serum (NHS) containing serial dilutions of 7A12 or control anti-C5a mAb at 37°C for 1 hour. RBC lysis was determined by measuring absorbance at OD405 nm. As expected, the control anti-C5 mAb 2G1 inhibited NHS-mediated ovine erythrocyte lysis by 50% in a dose-dependent manner. On the other hand, mAb 7A12 at doses ranging from 0.975 to 120 μg/ml did not exhibit 50% inhibition of NHS-mediated lysis of ovine erythrocytes.

In fig. 3, including FIG. 3A and 3B show results showing that, unlike the control anti-C5 mAb 2G1, which does not bind C5a, 7A12 binds human C5a in a dose-dependent manner, but it does not bind murine C5a. The plate was coated with human C5 or mouse C5. After incubation with serial dilutions of 7A12 or control anti-C5 mAb, bound mAb was detected using HRP-conjugated rabbit anti-mouse IgG. In fig. 3A shows that mAb 7A12 exhibited high reactivity to human C5a. Since mAb 7A12 reacts with the complete C5 protein (as shown in Fig. 1), it can be concluded that mAb 7A12 binds both the C5a fragment of native human C5 and free human C5a. On the other hand, control anti-C5a mAb was not observed to bind to human C5a. In fig. 3B shows that mAb 7A12 binding to C5a was specific to human C5a with little binding to mouse C5a.

In fig. 4, including FIG. 4A and 4B show the results of experiments assessing the binding affinity of mAb 7A12 to human C5 and C5a. Purified human C5 or C5a was coupled onto the CM4 chip using an amine coupling method. Biacore analysis was performed on a Biacore-2000 instrument. Between each binding, the chip was regenerated using 50 mM NaOH. mAb 7A12 binds to human C5 as shown in FIG. 4A, and human C5a, as shown in FIG. 4B, with the same affinity.

In fig. 5 shows results showing that mAb 7A12, but not the control anti-C5 mAb 2G1, inhibits C5a-mediated neutrophil migration. Human C5a at a concentration of 10 nM was used to induce chemotaxis of a human monocytic cell line, U937, transfected with the human C5a receptor. Cells were placed in the upper chamber of the Transwell system in the presence of mAb 7A12 or control anti-C5 mAb 2G1, and cell migration was quantified by counting cells in the lower chamber. mAb 7A12 showed complete inhibition of C5a-induced chemotaxis at a concentration of 10 μg/ml, whereas the control anti-C5 mAb 2G1 could not block C5a-induced chemotaxis.

In fig. 6, including FIG. 6A-6D show results showing that mAb 7A12, but not the control anti-C5 mAb 2G1, inhibits C5a-induced intracellular calcium mobilization in U937 cells. Calcium mobilization was absent in U937 cells expressing the human C5a receptor (U937-C5aR) in the absence of human C5a stimulation, as shown in FIG. 6A. Treatment with C5a (10 nM) resulted in a transient calcium influx in U937-C5aR cells, as shown in Fig. 6B, which could be inhibited by preincubation with mAb 7A12 (50 μg/ml) as shown in FIG. 6C, but not with the control anti-C5 mAb 2G1 (50 μg/ml) as shown in FIG. 6D. Arrows indicate the time point at which C5a or a mixture of C5a and antibody was added to the cell suspension.

In fig. 7, including FIG. 7A and 7B show the sequences of the heavy and light chain variable regions of mAb 7A12. In fig. 7A shows the nucleic acid sequences and amino acid sequences of VH mAb 7A12. The signal peptide is underlined and the sequences CDR1, CDR2 and CDR3 are highlighted in bold and grey. In fig. 7B shows the nucleic acid sequences and amino acid sequences of VL mAb 7A12. The signal peptide is underlined and the sequences CDR1, CDR2 and CDR3 are highlighted in bold and grey.

Detailed Description of the Invention

The present invention relates to inhibition of the complement signaling pathway using an anti-C5a antibody. In various embodiments, the invention provides compositions and methods for treating a complement-mediated disease or complement-mediated disorder in an individual by contacting the individual with an anti-C5a antibody. Complement-mediated diseases and disorders that can be treated with the compositions and methods of the invention include, but are not limited to, macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke , postoperative systemic inflammatory response syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH) syndrome, autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, neuromyelitis optica, NMO, multiple sclerosis, delayed graft function, antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS), central retinal vein occlusion (CRVO), central retinal artery occlusion (CRAO), epidermolysis bullosa, sepsis, septic shock, organ transplantation, inflammation (including but not limited to inflammation associated with cardiopulmonary bypass and renal dialysis), C3 glomerulopathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (including, but not limited to, antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, lupus nephritis and combinations thereof), ANCA - Shiga toxin-associated HUS vasculitis and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), or any combination thereof.

Definitions.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, illustrative methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The terms inhibit and inhibit as used herein mean reducing, suppressing, decreasing or blocking an activity or function by at least about 10% relative to a control value. In some embodiments, activity is inhibited or blocked by at least about 50% of the control value. In some embodiments, the activity is inhibited or blocked by at least about 75%. In some embodiments, the activity is inhibited or blocked by at least about 95%.

The terms effective amount and pharmaceutically effective amount refer to an amount of agent sufficient to provide the desired biological result. Such a result may be a reduction and/or alleviation of signs, symptoms or causes of a disease or disorder, or any other desired change in a biological system. The appropriate effective amount in any particular case can be determined by one skilled in the art using routine experimentation.

Terms patient, individual, individual, etc. are used interchangeably herein to refer to any animal, in some embodiments a mammal, and in some embodiments a human, having a complement system, including a human in need of treatment or susceptible to developing a condition or its complications. The individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, mice and humans.

The term abnormal, when used in the context of organisms, tissues, cells, or components thereof, refers to those organisms, tissues, cells, or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) .d.) from those organisms, tissues, cells or their components that exhibit a normal (expected/homeostatic) corresponding characteristic. Characteristics that are normal or expected in one cell, tissue type, or individual may be abnormal in another cell or tissue type.

A disease is a state of health of an individual in which the individual is unable to maintain homeostasis and in which, if the disease is not abated, the individual's health continues to deteriorate.

In contrast, a disorder in an individual represents a state of health in which the individual can maintain homeostasis, but the individual's health status is less favorable than it would be in the absence of the disorder. Without treatment, the disorder does not necessarily cause further deterioration in the individual's health.

A disease or disorder is alleviated if the severity of a sign or symptom of the disease or disorder, the frequency with which such sign or symptom, or both, experienced by the patient decreases.

An effective amount or therapeutically effective amount of a compound is that amount of the compound that is sufficient to provide a beneficial effect to the individual to whom the compound is administered.

As used herein, the term instructional material includes a publication, record, diagram, or any other medium of expression that can be used to communicate the suitability of a compound, composition, vector, or delivery system of the invention in a kit for alleviating various diseases or disorders identified in this document. Optionally or alternatively, the instructional material may describe one or more methods for alleviating diseases or disorders in a cell or tissue of a mammal. The instructional material of a kit of the invention may, for example, be attached to a container that contains a specified compound, composition, vector, or delivery system of the invention, or shipped with a container that contains a specified compound, composition, vector, or delivery system. Alternatively, the instructional material may be sent separately from the container with the intention that the instructional material and the connection will be used together by the recipient.

As used herein, the term operably linked or operatively linked can mean that the expression of a gene is under the control of a promoter to which it is spatially linked. A promoter can be located 5' (upstream) or 3' (downstream) of the gene that is under its control. The distance between a promoter and a gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, a change in this distance can be adapted without loss of promoter function.

Therapeutic treatment is treatment given to an individual exhibiting signs of a disease or disorder with the goal of reducing or eliminating those signs.

As used herein, the term treating a disease or disorder means reducing the incidence and/or severity of a sign and/or symptom of a disease or disorder from which a patient suffers.

As used herein, the expression biological specimen, specimen, or sample is intended to include any sample containing a cell, tissue, or body fluid in which expression of a nucleic acid or polypeptide can be detected. The biological sample may include any biological material suitable for detecting the desired biomarkers, and may contain cellular and/or non-cellular material obtained from the individual. Examples of such biological samples include, but are not limited to, blood, lymph, bone marrow, biopsy, and smears. Samples that are liquid in nature are referred to herein as biological fluids. Biological samples can be obtained from the patient in a variety of ways, including, for example, by scraping or swabbing an area or using a needle to obtain body fluids. Methods for collecting various body samples are well known in the art.

As used herein, the term antibody refers to an immunoglobulin molecule that is capable of specifically binding to a specific epitope of an antigen. Antibodies can be intact immunoglobulins obtained from natural sources or from recombinant sources, and can be immunoreactive fragments of intact immunoglobulins. Antibodies in the present invention can be in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (intrabodies), Fv, Fab, Fab', F(ab)2 and F(ab')2, and also single chain antibodies (scFv), heavy chain antibodies such as camel antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci., USA, 85:5879-5883; Bird et al., 1988, Science, 242:423-426).

As used herein, the term synthetic antibody means an antibody that is produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be interpreted to mean an antibody that has been produced by synthesizing a DNA molecule encoding an antibody, and such DNA molecule expresses an antibody protein or an amino acid sequence defining an antibody, wherein the DNA sequence or amino acid sequence was produced using synthetic DNA or amino acid sequence technology which is affordable and good

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As used herein, the term heavy chain antibody or heavy chain antibodies includes immunoglobulin molecules obtained from camelid species, either by immunization with a peptide and subsequent isolation of sera, or by cloning and expression of nucleic acid sequences encoding such antibodies. The term heavy chain antibody or heavy chain antibodies also covers immunoglobulin molecules isolated from an individual with a heavy chain disease, or obtained by cloning and expressing VH (immunoglobulin heavy chain variable region) genes from an individual.

A chimeric antibody refers to a type of engineered antibody that contains a naturally occurring variable region (light chain and heavy chain) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody.

A humanized antibody refers to a type of engineered antibody having CDRs derived from a non-human donor immunoglobulin and the remaining portions of the immunoglobulin-derived molecule derived from one (or more) human immunoglobulin(s). In addition, framework region residues can be modified to maintain binding affinity (see, for example, 1989, Queen et al., Proc. Natl. Acad Sci USA, 86:10029-10032; 1991, Hodgson et al., Bio/Technology , 9:421). A suitable human acceptor antibody may be an antibody that is selected from a conventional database, such as the KABAT database, the Los Alamos database, and the Swiss Protein database, based on homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody having homology to the framework regions of a donor antibody (amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable region framework for insertion of donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions can be selected in a similar manner. It should be noted that the heavy and light chains of the acceptor antibody do not necessarily originate from the same acceptor antibody. The prior art describes several methods for producing such humanized antibodies (see, for example, EP-A-0239400 and EP-A-054951).

The term donor antibody refers to an antibody (monoclonal and/or recombinant) that donates the amino acid sequences of its variable regions, CDRs or other functional fragments, or analogs thereof, to a first immunoglobulin partner to provide an altered immunoglobulin coding region, and the resulting altered antibody with antigenic expression expressed specificity and neutralizing activity characteristic of the donor antibody.

The term acceptor antibody refers to an antibody (monoclonal and/or recombinant) heterologous to a donor antibody that donates all (or any portion, but in some embodiments all) of the amino acid sequences constituting its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner. In certain embodiments, the human antibody is an acceptor antibody.

CDRs are defined as the complementarity determining regions of the amino acid sequences of an antibody, which are the hypervariable regions of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S., Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the immunoglobulin variable region. The structure and folding of the antibody protein may mean that other residues are considered part of the antigen-binding region, and should be understood by one skilled in the art as such. See, for example, Chothia et al. (1989), Conformations of immunoglobulin hypervariable regions, Nature, 342, p. 877-883. One of ordinary skill in the art will appreciate that there are many methods and techniques used to predict CDR sequences. Thus, the CDR sequences of a particular antibody may vary somewhat depending on what methods and techniques are used to predict the CDR sequences. Exemplary methods and techniques include, but are not limited to, those described by Lyskov et al., 2013, PLoS One, 8(5):e63906; Kunik, et al., 2012, Nucleic. Acids. Res., 40:W521-524; Marcatili et al., 2008, Bioinformatics, 24:1953; Chothia et al., 1989, Nature, 342:887; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD; Johnson et al., 2000, Nucleic. Acids. Res. 28:214; Martin et al., 1989, P.N.A.S. 86:9268; MacCallum et al., 1996, J. Mol. Biol., 5:732; and Dunbar et al., 2016, Nucleic Acids Res. 44:W474-478.

As used herein, the term immunoassay refers to any binding assay that uses an antibody capable of specifically binding to a target molecule to detect and quantify the target molecule.

As used herein, the term specifically binds with respect to an antibody means an antibody that recognizes and binds to a specific target molecule, but does not substantially recognize or bind to other molecules in the sample. In some cases the terms

- 6 043843 specific binding or binding is specifically used to mean that recognition and binding depend on the presence of a particular structure (eg, antigenic determinant or epitope) on the target molecule. If, for example, an antibody specifically binds to epitope A, then the presence of an unlabeled molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled A and antibody will result in a decrease in the amount of labeled A bound to the antibody.

The coding region of a gene consists of nucleotide residues of the coding strand of the gene and nucleotides of the non-coding strand of the gene that are homologous or complementary, respectively, to the coding region of the mRNA molecule that is synthesized by transcription of the gene.

The coding region of an mRNA molecule also consists of nucleotide residues of the mRNA molecule that correspond to the anticodon region of the transfer RNA molecule during translation of the mRNA molecule or that encode a stop codon. Thus, the coding region may include nucleotide residues containing codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (eg, amino acid residues in a protein export signal sequence).

Differentially reduced expression or down regulation refers to levels of a biomarker product that are at least 10% or more, for example 20, 30, 40 or 50, 60, 70, 80, 90% less or less and/or 2.0 , 1.8, 1.6, 1.4, 1.2, 1.1 times lower or less, and any and all full or partial intervals between them, than the control.

Differentially increased expression or up-regulation refers to levels of a biomarker product that are at least 10% or more, for example 20, 30, 40 or 50, 60, 70, 80, 90% higher or more and/or 1.1 , 1.2, 1.4, 1.6, 1.8, 2.0 times higher or more, and any and all full or partial intervals between them, than the control.

The term complementary, as used in relation to nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (base pairing) with a residue of a second nucleic acid region that is antiparallel to the first region if the residue is thymine or uracil. Likewise, it is also known that a cytosine residue of the first strand of a nucleic acid is capable of base pairing with a residue of the second strand of a nucleic acid that is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are antiparallel, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In some embodiments, the first region comprises a first region and the second region contains a second region, wherein when the first and second regions are antiparallel, at least about 50%, or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues in the first region are capable of base pairing with nucleotide residues in the second region. In some embodiments, all nucleotide residues in the first region are capable of pairing with nucleotide residues in the second region.

As used herein, the term DNA is defined as deoxyribonucleic acid.

Coding refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes having either a specific nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or specific amino acid sequence, and provide the resulting biological properties. Thus, a gene encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence, and is usually indicated in sequence listings, and the non-coding strand, used as a template for transcription of a gene or cDNA, may be referred to as encoding a protein or other product of that gene or cDNA.

Unless otherwise indicated, a nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate variants of each other and that encode the same amino acid sequence. The expression nucleotide sequence that encodes a protein or RNA may also include introns to the extent that the nucleotide sequence that encodes a protein may in some embodiment contain intron(s).

The term hybridoma as used herein refers to a cell resulting from the fusion of a B lymphocyte and a fusion partner, such as a myeloma cell. The hybridoma can be cloned and maintained indefinitely in cell culture and is capable of producing monoclonal antibodies. A hybridoma can also be considered a hybrid cell.

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Isolated means altered or removed from the natural state. For example, a nucleic acid or peptide present in a normal context in a living individual in nature is not isolated, but the same nucleic acid or peptide, partially or completely separated from coexisting substances in its natural environment, is isolated. The isolated nucleic acid or protein may be in substantially purified form or may be in a non-natural environment, such as, for example, a host cell.

Isolated nucleic acid refers to a segment or fragment of nucleic acid that has been separated from the sequences flanking it in its natural state, i.e. a DNA fragment that has been removed from sequences that are normally adjacent to the fragment, i.e. sequences adjacent to a fragment in the genome in which it occurs naturally. The term is also used to refer to nucleic acids that are substantially purified from other components that accompany the nucleic acid in nature, i.e. RNA or DNA, or proteins that accompany it in a cell in nature. Thus, the term includes, for example, recombinant DNA that has been incorporated into a vector, an autonomously replicating plasmid or virus, or the genomic DNA of a prokaryote or eukaryote, or that is present as a single molecule (i.e., as a cDNA or genomic , or a cDNA fragment obtained by PCR or restriction enzyme digestion) independent of other sequences. It also includes recombinant DNA, which is part of a hybrid gene encoding an additional polypeptide sequence.

In the context of the present invention, the following abbreviations for the most commonly occurring nucleic acid bases are used. A refers to adenosine, C refers to cytosine, G refers to guanosine, T refers to thymidine and U refers to uridine.

As used herein, the term polynucleotide is defined as a chain of nucleotides. Moreover, nucleic acids are polymers of nucleotides. Thus, the terms nucleic acids and polynucleotides as used herein are used interchangeably. One skilled in the art will recognize that nucleic acids are polynucleotides that can be hydrolyzed to monomeric nucleotides. Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences that are produced by any methods available in the art, including, without limitation, recombinant methods, i.e. cloning of nucleic acid sequences from a recombinant library or cellular genome, using conventional cloning and PCR technologies, etc. and using synthetic methods.

As used herein, the terms peptide, polypeptide and protein are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that a protein or peptide sequence may contain. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art, such as peptides, oligopeptides and oligomers, and longer chains, which are also commonly referred to in the art as proteins, of which there are many. types. Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

As used herein, the term progeny refers to an offspring or progeny and includes the progeny of a mammal and also includes a different or non-different progeny cell derived from a parent cell. In one usage, the term progeny refers to a descendant cell that is genetically identical to the parent. In other usage, the term progeny refers to a descendant cell that is genetically and phenotypically identical to the parent. In yet another usage, the term progeny refers to a descendant cell that was different from the parent cell.

As used herein, the term RNA is defined as ribonucleic acid.

As used herein, the term recombinant DNA is defined as DNA obtained by combining DNA fragments from different sources.

As used herein, the term recombinant polypeptide is defined as a polypeptide produced using recombinant DNA techniques.

As used herein, the term conjugated refers to the covalent attachment of one molecule to a second molecule.

Variant as a term used herein is a nucleic acid sequence or peptide sequence that differs from the reference

- 8 043843 nucleic acid sequence or peptide sequence, respectively, but retains the basic biological properties of the reference molecule. Changes in the sequence of a variant nucleic acid may not result in a change in the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of variant peptides are usually limited or conservative, such that the sequences of the reference peptide and the variant are generally very similar and identical in many areas. The variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The nucleic acid or peptide variant may be naturally occurring, such as an allelic variant, or may represent a variant that is not known to occur in nature. Non-naturally occurring variants of nucleic acids and peptides can be produced by mutagenesis or direct synthesis. In various embodiments, the sequence is at least 99% variant, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85% identical to the reference sequence.

As used herein, the term regulation can mean any method of changing the level or activity of a substrate. Non-limiting examples of protein regulation include effects on expression (including transcription and/or translation), effects on folding, effects on protein degradation or metabolism, and effects on protein localization. Non-limiting examples of regulation on an enzyme further include effects on enzymatic activity. A regulator refers to a molecule whose activity involves influencing the level or activity of a substrate. The regulator can be direct or indirect. A regulator may function to activate or inhibit or otherwise modulate its substrate.

As used herein, the term scan window refers to a segment of a number of contiguous positions at which a sequence can be evaluated independently of any flanking sequence. The scanning window typically shifts gradually along the length of the sequence to be scored, with each new segment scored independently. The gradual shift can be 1 or more than one position.

As used herein, the term vector can mean a nucleic acid sequence containing an origin of replication. The vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA based vector. The vector can be either a self-replicating extrachromosomal vector or a vector that integrates into the host genome.

Ranges: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as strictly limiting the scope of the invention.

Accordingly, the range description should be considered to specifically disclose all possible subranges as well as the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6 etc., as well as individual numbers in this range, for example 1; 2; 2.7; 3; 4; 5; 5,3 and 6. This applies regardless of the band width.

Description.

The present invention relates to inhibition of the signaling pathway and complement-related diseases and disorders using an anti-C5a antibody. In one embodiment, the invention is directed to inhibiting the complement signaling cascade by specifically targeting the complement component C5 and its cleavage product C5a protein, while the C5 cleavage product C5b remains functional. In one embodiment, the invention is directed to methods for treating and preventing inflammation and autoimmune diseases and disorders mediated by unwanted, uncontrolled or excessive complement activation. In one embodiment, the invention is directed to treating a complement-mediated disease or complement-mediated disorder in an individual by exposing the individual to an anti-C5a antibody. In some embodiments, the invention provides methods for treating C5a-mediated chemotaxis while leaving C5b-mediated MAC assembly intact.

In one embodiment, the invention provides a method of treating a complement-mediated disease or disorder in an individual, comprising the step of administering to said individual an anti-C5a antibody, thereby selectively inhibiting the effects of the C5a protein. Examples of complement-mediated diseases and disorders that can be treated using the methods of the invention include, but are not limited to, macular degeneration (MD), age-related macular

- 9 043843 ny degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, postoperative systemic inflammatory response syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria syndrome ( PNH), autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, neuromyelitis optica, NMO, multiple sclerosis, delayed graft function, antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS), central retinal vein occlusion (CRVO) , central retinal artery occlusion (CRAO), epidermolysis bullosa, sepsis, septic shock, organ transplantation, inflammation (including but not limited to inflammation associated with cardiopulmonary bypass and renal dialysis), C3 glomerulopathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (including, but not limited to, antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, lupus nephritis, and combinations thereof), ANCA-associated Shiga toxin HUS vasculitis, and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), or any combinations of them. In some embodiments, the AR-mediated disease is sepsis, septic shock, rheumatoid arthritis, autoimmune hemolytic anemia, GvHD, antiphospholipid syndrome, or Gaucher disease.

The ability of the immune system to distinguish between self and non-self antigens is vital to the functioning of the immune system as a specific defense against invading microorganisms. Non-self antigens represent antigens that are part of substances entering or present in the body that are noticeably different or foreign compared to the individual’s own components, while self antigens represent antigens that are not noticeably different in a healthy individual or are not foreign compared to their own components. In various embodiments of the methods, the complement activation that is inhibited is the activation that was initiated by at least one of the group consisting of a microbial antigen, a non-biological foreign surface, altered self tissue, or combinations thereof. One example of a non-biological foreign surface is tubing for blood vessels, such as those used in cardiopulmonary bypass or kidney dialysis. Examples of altered self tissues include apoptotic, necrotic and ischemic tissues and cells, or combinations thereof.

In some embodiments, the anti-C5a antibodies of the invention inhibit the downstream effects of activation of the alternative complement pathway (AP), the classical pathway (CP), or the lectin pathway (LP). Typically, SR is initiated by antigen-antibody complexes, LP is activated by the binding of lectins to sugar molecules on the surfaces of microorganisms, while AR is constitutively active at a low level, but can be rapidly upregulated on the surfaces of bacterial, viral and parasitic cells due to the absence of regulatory proteins. Host cells are usually protected from AR activation of complement by regulatory proteins. But in some situations, such as when regulatory proteins are defective or missing, the AR can also be activated uncontrollably on host cells, leading to the development of a complement-mediated disease or disorder. SR consists of components C1, C2, C4 and converges with AR at the activation stage of C3. LP is composed of mannose-binding lectins (MBLs) and MBL-associated serine proteases (MASPs) and shares components C4 and C2 with CP. AR consists of components C3 and several factors such as factor B, factor D, roperdine, C5 and factor H, a regulator in the liquid phase. Complement activation consists of three stages:

(a) recognition;

(b) enzyme activation; and (c) membrane attack leading to cell death.

The first phase of complement CP activation begins with C1. C1 consists of three distinct proteins: a recognition subunit, C1q, and serine protease subcomponents, C1r and C1s, which are linked together in a calcium-dependent tetrameric complex, C1r2s2. An intact C1 complex is required for physiological activation of C1. Activation occurs when the intact C1 complex binds to immunoglobulin complexed with the antigen. This binding activates C1s, which then cleaves the C4 and C2 proteins to form C4a and C4b, as well as C2a and C2b. The C4b and C2a fragments combine to form the C3 convertase, C4b2a, which in turn cleaves C3 to form C3a and C3b. LP activation is initiated by the binding of MBL to certain sugars on the target surface, and this triggers the activation of MBL-associated serine proteases (MASPs), which then cleave C4 and C2 in a manner similar to the activity of C1s in the CP pathway, leading to the generation of the C3 convertase, C4b2a. Thus, CP and LP are activated by different mechanisms, but they share the same C4 and C2 components, and both pathways result in the same C3 convertase, C4b2a. The cleavage of C3 by C4b2a into C3b and C3a is a central event in the complement pathway for two reasons. It initiates the AP amplification loop, since C3b deposited on the surface is the central intermediate product of AP. Both C3a and C3b are biologically important. C3a is pro-inflammatory and, together with C5a, is an anaphylatoxin. C3b and further products of its cleavage also bind to complement receptors located on neutrophils, eosinophils, monocytes and macrophages, thereby promoting phagocytosis and clearance of C3b-opsonized particles. Finally, C3b can bind to C4b2a to form the C5 convertase CP and LP to activate the terminal complement sequence, resulting in the formation of C5a, a potent proinflammatory mediator, and the assembly of the lytic membrane attack complex (MAC), C5-C9.

In one embodiment, the complement pathway activity that is inhibited using the method of the invention is complement pathway activation induced by at least one from the group selected from lipopolysaccharide (LPS), lipooligosaccharide (LOS), pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). In another embodiment, the activity of the complement signaling pathway that is inhibited using the method of the invention is the activity of the C5a protein. In yet another embodiment, the activity of the complement pathway that is inhibited using the method of the invention is C5a dependent.

In one embodiment, the invention provides a method of inhibiting the initiation of an inflammatory cascade through activation of terminal complement in an individual, comprising the step of administering to said individual an anti-C5a antibody, thereby inhibiting the initiation of C5a-dependent inflammation through activation of terminal complement resulting from CP activation, LP or AP of an individual. Examples of these embodiments are patients with sepsis who suffer from complement-mediated systemic inflammation, and individuals suffering from conditions that may be caused by complement-mediated organ-specific inflammation, such as aHUS, AIHA, antiphospholipid syndrome, GVHD, asthma, ischemia/reperfusion injury, rheumatoid arthritis and ANCA-mediated kidney diseases. In various embodiments, diseases and disorders that can be treated using the compositions and methods of the invention include, but are not limited to, complement-mediated hemolysis, complement-mediated aHUS, C3 glomerulopathy, neuromyelitis optica, myasthenia gravis, asthma, ischemia/reperfusion injury, sepsis, septic shock, rheumatoid arthritis and ANCA-mediated renal diseases or disorders. In some embodiments, the AR-mediated disease is sepsis, septic shock, rheumatoid arthritis, autoimmune hemolytic anemia, GvHD, antiphospholipid syndrome, or Gaucher disease.

In various other embodiments, methods are provided herein for identifying a potential anti-C5a antibody having an inhibitory effect on the complement signaling pathway. One such method includes the following steps:

a) stable transfection of cells with the C5a receptor;

b) seeding cells in chemotaxis assay buffer (RPMI medium with 0.5% BSA) into the upper chambers of Transwell inserts;

c) adding 10 nM recombinant human complement C5a, pretreated with antibody, to the chemotaxis assay buffer;

c) incubation for 3 hours at 37°C;

d) collecting migrated cells from the lower chambers and counting cells using a Coulter counter;

f) comparing the number of cells seeded in relation to those migrating into the lower chamber of the Transwell, which received recombinant human complement C5a, with the number of cells migrating into the lower chamber with a positive control in the Transwell system and with a negative control in the Transwell system, where, as the number of migrated cells decreases compared to the positive control, an anti-C5a antibody is identified.

In various other embodiments, methods are provided herein for identifying a potential anti-C5a antibody having an inhibitory effect on the complement signaling pathway. One such method includes the following steps:

a) stable transfection of cells with the C5a receptor;

b) washing cells with HEPES buffer for calcium mobilization assay (25 mM HEPES, 119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl ₂ and 1 mM CaCl ₂ ) containing 1 mg/ml bovine serum albumin (BSA);

c) incubating cells with 1 μM indo-1-acetoxymethyl ether at room temperature for 30 minutes;

d) washing the cells twice and resuspending in 1.3 ml of the above buffer;

f) mixing 10 nM human C5a protein and 50 μg/ml anti-C5a antibody to a final concentration of 10 nM protein and 50 μg/ml antibody at room temperature;

f) measurement of intercellular Ca ²⁺ at an excitation wavelength of 360 nm and an emission wavelength of 415 nm using an Infinite F200 multi-mode microplate reader;

g) comparison of C5a-induced intercellular calcium mobilization in cells treated with a positive control and a negative control, where when calcium mobilization is reduced compared to the positive control, an anti-C5a antibody is identified.

- 11 043843

Anti-C 5a antibodies.

In some embodiments, the invention includes compositions containing an antibody that specifically binds to C5 and C5a. In one embodiment, the anti-C5a antibody of the invention specifically binds to C5. In one embodiment, the anti-C5a antibody of the invention specifically binds to C5a. In one embodiment, the anti-C5a antibody of the invention specifically binds to both C5 and C5a. In one embodiment, the anti-C5a antibody of the invention specifically binds to both the C5a fragment of C5 and free C5a. In some embodiments, the anti-C5a antibody of the invention specifically binds to C5 but does not block the cleavage of C5 into C5a and C5b. In one embodiment, the anti-C5 antibody is a polyclonal antibody. In yet another embodiment, the anti-C5 antibody is a monoclonal antibody. In some embodiments, the anti-C5 antibody does not block the cleavage of C5 into C5a and C5b, but does inhibit C5a-dependent biological activity. In some embodiments, the anti-C5a antibody is a chimeric antibody. In yet other embodiments, the anti-C5a antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, C5a is human C5a.

In some embodiments, binding of an antibody or antibody fragment to human C5a is associated with a decrease in the expression level or half-life of C5a in the intact organism. In some embodiments, the invention provides a protein or polypeptide capable of binding to human C5a. In some embodiments, the antibody or antibody fragment, protein or polypeptide binds to a corresponding region or fragment or epitope of human C5a, and binding of the antibody or antibody fragment or protein or polypeptide to the corresponding region of human C5a is associated with a decrease in expression level or half-life C5a in an intact organism.

In some embodiments, binding of an antibody or antibody fragment to human C5a is associated with a decrease in C5a activity in the complement pathway in the intact organism. In some embodiments, the invention provides a protein or polypeptide capable of binding to human C5a. In some embodiments, the antibody or antibody fragment, protein or polypeptide binds to a corresponding region or fragment or epitope of human C5a; and binding of an antibody, or antibody fragment, or protein, or polypeptide to the corresponding site of human C5a is associated with a decrease in C5a activity in the intact organism.

In some embodiments, a human C5a binding antibody or C5a binding antibody fragment is further conjugated to a protein, peptide, or other compound. In some embodiments, a C5a-binding antibody or antibody fragment is conjugated to a protein, peptide, or other compound. In some embodiments, the protein, peptide, or other compound to which the C5-binding antibody or antibody fragment is conjugated is a targeting molecule (ie, the targeting molecule specifically binds to a molecule other than C5a). In some embodiments, the protein, peptide, or other compound to which the C5a-binding antibody or antibody fragment is conjugated is an effector molecule (eg, a cytotoxic molecule).

In one embodiment, the anti-C5a antibody or antigen binding fragment thereof comprises at least one of CDRs selected from the group consisting of

V H-CDR1: SEQ ID NO: 3;

V H-CDR2: SEQ ID NO: 4;

V H-CDR3: SEQ ID NO: 5;

V L-CDR1 : SEQ ID NO: 8;

V L-CDR2: SEQ ID NO: 9; And

VL-CDR3: SEQ ID NO: 10, or a variant or variants thereof.

In yet another embodiment, the anti-C5 antibody comprises all CDRs from the group consisting of

V H-CDR1: SEQ ID NO: 3;

V H-CDR2: SEQ ID NO: 4;

V H-CDR3: SEQ ID NO: 5;

VL-CDR1: SEQ ID NO: 8;

VL-CDR2: SEQ ID NO: 9; And

VL-CDR3: SEQ ID NO: 10, or a variant or variants thereof.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises

VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions; And

- 12 043843

VL-CDR1: SEQ ID NO: 8 or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises

VH-CDR1 containing the amino acid sequence SEQ ID NO: 3; And

VL-CDR1: SEQ ID NO: 8.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises

VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions; And

VL-CDR2: SEQ ID NO: 9 or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises

VH-CDR2 containing the amino acid sequence SEQ ID NO: 4; And

VL-CDR2: SEQ ID NO: 9.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises

VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions; And

VL-CDR3: SEQ ID NO: 10 or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises

VH-CDR3 containing the amino acid sequence SEQ ID NO: 5; And

VL-CDR3: SEQ ID NO: 10.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises

V H-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions;

V H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions;

V H-CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions;

V L-CDR1 containing the amino acid sequence of SEQ ID NO: 8, or a variant thereof containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions;

V L-CDR2 containing the amino acid sequence of SEQ ID NO: 9, or a variant thereof containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions; And

VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises

V H-CDR1 containing the amino acid sequence of SEQ ID NO: 3, or a variant thereof containing up to about 3 (in particular, any of 1, 2 or 3) amino acid substitutions;

V H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof containing up to about 3 (especially about 1, 2 or 3) amino acid substitutions;

V H-CDR3 containing the amino acid sequence SEQ ID NO: 5;

V L-CDR1 containing the amino acid sequence of SEQ ID NO: 8, or a variant thereof containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions;

V L-CDR2 containing the amino acid sequence of SEQ ID NO: 9, or a variant thereof containing up to about 3 (in particular, about 1, 2 or 3) amino acid substitutions; And

V L-CDR3 containing the amino acid sequence SEQ ID NO: 10.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises

V H-CDR1 containing the amino acid sequence SEQ ID NO: 3;

V H-CDR2 containing the amino acid sequence SEQ ID NO: 4;

V H-CDR3 containing the amino acid sequence SEQ ID NO: 5;

V L-CDR1 containing the amino acid sequence SEQ ID NO: 8;

V L-CDR2 containing the amino acid sequence SEQ ID NO: 9; And

V L-CDR3 containing the amino acid sequence SEQ ID NO: 10.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof. In other embodiments, the anti-C5a antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof. In yet another embodiment, the anti-C5a antibody is a monoclonal antibody designated mAb 7A12. The monoclonal anti-C5a antibody, designated mAb 7A12, contains a heavy chain containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof, and a light chain containing the amino acid sequence of SEQ ID NO: 7 or a variant thereof. In some embodiments, the anti-C5a monoclonal antibody is a humanized antibody having one or more or all of the CDRs or variants thereof, a mAb designated 7A12. In some embodiments, the anti-C5a monoclonal antibody is a chimeric antibody having one or more or all of the CDRs or variants thereof, a mAb designated 7A12.

In some embodiments, the anti-C5a antibody or antigen binding fragment thereof comprises at least one of a heavy chain CDR1, CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof. In other embodiments, the anti-C5 antibody or antigen binding fragment thereof comprises at least one of a light chain CDR1, CDR2 and CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof.

In some embodiments, the antibodies are chimeric antibodies. In some embodiments, an anti-human C5a antibody may comprise human light chain and human heavy chain constant regions in combination with variable region CDR sequences or a variant thereof described elsewhere herein. One skilled in the art can prepare and produce a chimeric antibody using known techniques for replacing the appropriate domains of the specific antibodies of interest. Such an antibody is readily prepared by grafting heterogeneous antibody domains by incorporating one or more of the CDR sequences described herein. Using known recombinant technology, a recombinant antibody containing heavy and light chain constant regions encoded by human heavy and light chain constant region nucleic acid sequences can be prepared and prepared; and heavy and light chain variable regions containing CDRs encoded by nucleic acid sequences corresponding to the CDR sequences given in the disclosure. One skilled in the art can prepare an anti-human C5a antibody containing one or more of the CDR sequences described in this disclosure, wherein regions of one light chain or regions of one heavy chain are replaced by regions from an antibody of another species, such as a human. A human anti-human C5a antibody comprising variable regions having one or more CDR sequences selected from SEQ ID NOs: 3-5 and 8-10, or a variant or variants thereof, in combination with mouse or non-mouse antibody structural elements outside the CDR region , can be obtained by conventional methods known in the art. In some embodiments, the antibodies or antibody fragments are further humanized using techniques known in the art.

In some embodiments, an anti-C5a antibody includes an antibody having at least about 85% amino acid sequence identity to one or more of the CDR sequences described herein shown in SEQ ID NOs: 3-5 and 8-10.

In one embodiment, the invention provides an anti-C5a antibody having CDR sequences at least about 85% identical to the CDR sequences described herein. The invention covers an anti-C5a antibody or antigen binding fragment thereof having CDR sequences that are at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% identical to the CDR sequences described herein. In one embodiment, the anti-human C5a antibody has a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region has an amino acid sequence that is at least about 85% identical to the sequence of SEQ ID NO: 2, and wherein the VL region has an amino acid sequence that is at least about 90% identical to the sequence of SEQ ID NO: 7. In some embodiments, the antibody or antibody fragment is modified. In some embodiments, the modifications include fusing an antibody or an antigen-binding fragment thereof with regions of another protein or protein fragment. In some embodiments, an antibody or antibody fragment of the invention is modified to increase its half-life in the bloodstream in vivo. For example, an antibody fragment can be fused to an FcRn molecule, which is also known as the neonatal Fc receptor, to stabilize the antibody in vivo (Nature Reviews Immunology, 7:715-725). One skilled in the art can prepare a human C5a-binding single chain variable fragment (scFv) containing at least one CDR sequence selected from SEQ ID NOs: 3-5 and 8-10. The scFv may comprise at least one heavy chain variable region sequence shown in SEQ ID NOs: 3-5 and at least one light chain variable region sequence shown in SEQ ID NOs: 8-10. CDR sequences included in scFv having amino acid sequence identity at 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99 or 100% with the CDR sequences described herein

- 14 043843 disclosure are included in the scope of this disclosure.

In some embodiments, the antibody or antibody fragment is modified. In some embodiments, the modifications include fusion of an antibody or an antigen-binding fragment thereof with regions of another protein or protein fragment. In some embodiments, an antibody or antibody fragment of the invention is modified to increase its half-life in the bloodstream in vivo. For example, an antibody fragment can be fused to an FcRn molecule, which is also known as the neonatal Fc receptor, to stabilize the antibody in vivo (Nature Reviews Immunology, 7:715-725). In some embodiments, the antibody or antigen-binding fragment thereof is conjugated (eg, fused) to an effector molecule and/or another targeting molecule (such as an antibody or antibody fragment that recognizes another molecule, another antigen, or another epitope).

In various embodiments, any of the antibodies of the invention described herein having any of the variable regions described herein may comprise a human IgG4 heavy chain constant region. In some embodiments, an antibody of the invention comprises a human IgG4 heavy chain constant region having the S228P mutation.

One skilled in the art can prepare a C5a-binding single chain variable fragment (scFv) containing at least one specific CDR sequence selected from SEQ ID NOs: 3-5, 8-10, or variant or variants thereof. The scFv may comprise the heavy chain variable region sequences shown in SEQ ID NO: 3-5, or a variant or variants thereof, and the light chain variable region sequences shown in SEQ ID NO: 8-10, or a variant or variants thereof. CDR sequences included in the scFv having at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% amino acid sequence identity with the CDR sequences described in the present disclosure are included within the scope of the present disclosure.

Screening tests.

The present invention finds use in various screening tests, including determining whether a candidate anti-C5a antibody can inhibit complement activity.

In some embodiments, the level of complement activity in the presence of a candidate anti-C5 antibody is compared with the complement activity established for a positive control. A positive control involves activation of complement in the absence of an added test compound or in the presence of another test compound that does not bind C5a. In some embodiments, a candidate anti-C5a antibody is identified as a complement inhibitor when the complement activity in the presence of the candidate anti-C5a antibody is less than about 70% of the complement activity detected in the positive control; this corresponds to more than about 30% inhibition of complement activity in the presence of the test compound. In yet other embodiments, a candidate anti-C5a antibody is identified as a complement inhibitor when the complement activity in the presence of the candidate anti-C5a antibody is below about 80% of the complement activity detected in the positive control; this corresponds to more than about 20% inhibition of complement activity in the presence of the test compound. In yet other embodiments, a candidate anti-C5a antibody is identified as a complement inhibitor when the complement activity in the presence of the candidate anti-C5a antibody is less than about 90% of the complement activity detected in the positive control; this corresponds to more than about 10% inhibition of complement activity in the presence of the test compound. In some embodiments, the level of complement inhibition of the candidate anti-C5a antibody is compared to the level of inhibition established in the negative control.

A variety of immunoassay formats, including competitive and noncompetitive immunoassay formats, antigen capture assays, two-antibody sandwich assays, and three-antibody sandwich assays, are suitable methods for the invention (Self et al., 1996, Curr. Opin. Biotechnol ., 7:60-65). The invention should not be construed as limited to any one type of known or currently unknown assay, provided that the assay is capable of detecting complement inhibition.

An enzyme-linked immunosorbent assay (ELISA) is suitable for use in the methods of the invention. An enzyme, such as, but not limited to, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase or urease, can be coupled, for example, to an antibody or secondary antibody for use in the method of the invention. The horseradish peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which in the presence of hydrogen peroxide gives a soluble product detectable at 450 nm. Other suitable enzyme coupled systems include, for example, an alkaline phosphatase detection system, which can be used with the chromogenic substrate p-nitrophenyl phosphate to produce a soluble product readily detectable at 405 nm. Similarly, the beta-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-beta-O-galactopyranoside

- 15 043843 (ONPG) to obtain a soluble product detectable at 410 nm. Alternatively, a urease detection system with a substrate such as urea-bromocresol purple (Sigma Immunochemicals, St. Louis, MO) can be used. Suitable enzyme-linked primary and secondary antibodies can be obtained from any number of commercial sources.

Chemiluminescent detection is also suitable for detecting complement inhibition. Chemiluminescent secondary antibodies can be obtained from any number of commercial sources.

Fluorescent detection is also useful for detecting complement inhibition. Suitable fluorochromes include, but are not limited to, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and antibodies labeled with lissamine, fluorescein, or rhodamine.

Radioimmunoassay (RIA) is also useful in the methods of the invention. Such assays are well known in the art and are described, for example, in Brophy et al. (1990, Biochem. Biophys. Res. Comm., 167:898-903) and Guechot et al. (1996, Clin. Chem., 42:558-563). Radioimmunoassay is carried out, for example, using a primary or secondary antibody labeled with iodine-125 (Harlow et al., supra, 1999).

The signal emitted by the detectable antibody is analyzed, for example, using a spectrophotometer to detect the color of the chromogenic substrate; radiation counter for detecting radiation, such as a gamma counter for determining iodine-125; or a fluorometer to analyze fluorescence in the presence of light of a specific wavelength. If an enzyme-linked assay is used, the quantitative assay is performed using a spectrophotometer. It is understood that the assays of the invention can be performed manually or, if desired, can be automated, and that the signal emitted from multiple samples can be detected simultaneously using many commercially available systems.

The methods of the invention also include the use of capillary electrophoresis immunoassays (CEIA), which can be automated if desired. Immunoassays can also be used in combination with laser-induced fluorescence, as described, for example, in the publications of Schmalzing et al. (1997, Electrophoresis, 18:2184-2193) and Bao (1997, J. Chromatogr. B. Biomed. Sci., 699:463-480). Liposomal immunoassays, such as flow injection liposomal immunoassays and liposomal immunosensors, can also be used in accordance with the methods of the invention (Rongen et al., 1997, J. Immunol. Methods, 204:105-133).

Quantitative Western blotting can also be used to determine the level of complement inhibition in the methods of the invention. Western blots are quantitatively analyzed using well-known methods such as scanning densitometry (Parra et al., 1998, J. Vase. Surg., 28:669-675).

Methods of administration.

The methods of the invention include administering a therapeutically effective amount of at least one anti-C5a antibody or antigen binding fragment thereof (such as any of the antibodies or fragments thereof described elsewhere herein) to an individual identified as having or suspected of having a complement-mediated disease or disorder. In one embodiment, the individual is a mammal having a complement system. In one embodiment, the individual represents a person. In various embodiments, at least one anti-C5a antibody or antigen binding fragment thereof is administered locally, regionally, or systemically.

In various embodiments, the disease or disorder is at least selected from the group consisting of macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, postoperative systemic inflammatory syndrome response, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH) syndrome, myasthenia gravis, neuromyelitis optica, NMO, multiple sclerosis, delayed graft function, antibody-mediated rejection, atypical hemolytic uremic syndrome ( aHUS), central retinal vein occlusion (CRVO), central retinal artery occlusion (CRAO), epidermolysis bullosa, sepsis, septic shock, organ transplantation, inflammation (including but not limited to inflammation associated with cardiopulmonary bypass and renal dialysis), C3- glomerulopathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (including, but not limited to, antineutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis, lupus nephritis, and combinations thereof), ANCA-associated vasculitis, Shiga toxin-induced HUS, and antiphospholipid antibody-induced wasting pregnancy, graft-versus-host disease (GVHD), or any combination thereof. In some embodiments, the AR-mediated disease is sepsis, septic shock, rheumatoid arthritis, autoimmune hemolytic anemia, GvHD, antiphospholipid syndrome, or Gaucher disease. The methods of the invention may include administration of at least one anti-C5a antibody or antigen binding fragment thereof, but the present invention should in no way be construed as limited to the anti-C5a antibodies described herein, but rather should be considered to cover any anti-C5a antibody, both known and unknown, which reduces and decreases complement activation.

The method of the invention includes administering a therapeutically effective amount of at least one anti-C5a antibody or antigen binding fragment thereof to an individual, wherein the composition of the present invention contains at least one anti-C5a antibody or antigen binding fragment thereof, alone or in combination of at least with one other therapeutic agent. The invention can be used in combination with other treatment methods, such as, for example, anti-inflammatory therapy and the like. Examples of anti-inflammatory therapy that can be used in combination with the methods of the invention include, for example, therapy that uses steroid drugs, as well as therapy that uses non-steroidal drugs.

The method of the invention includes administering to a subject a therapeutically effective amount of an anti-C5a antibody or an antigen-binding fragment thereof. In some embodiments, the invention provides a method of treating C5a-related diseases involving dysregulation of the complement signaling pathway by administering a therapeutically effective amount of an antibody of the invention or a therapeutically effective amount of an antibody fragment such that the result is a decrease in C5a activity in an individual. In some embodiments, the invention provides a method of treating C5a-related diseases involving dysregulation of the complement signaling pathway by administering a therapeutically effective amount of an antibody or antibody fragment. In some embodiments, the invention provides a method of treating C5a-related diseases involving dysregulation of the complement signaling pathway by administering to an individual an effective amount of an antibody, antibody fragment, polypeptide, peptide, or conjugated peptide such that complement activity is reduced in the individual. In some embodiments, the treatment method comprises systemically administering to an individual an effective dose of an antibody or antibody fragment, whereby the individual experiences a systemic reduction in C5a activity.

Administration of an anti-C5a antibody or an antigen-binding fragment thereof in the treatment method of the invention can be achieved in a variety of ways using methods known in the art. Thus, the therapeutic and prophylactic methods of the invention include the use of pharmaceutical compositions containing an anti-C5a antibody.

Pharmaceutical compositions suitable for the practice of the invention can be administered to deliver a dose of at least about 1 ng/kg, at least about 5 ng/kg, at least about 10 ng/kg, at least about 25 ng/kg. kg, at least about 50 ng/kg, at least about 100 ng/kg, at least about 500 ng/kg, at least about 1 μg/kg, at least about 5 μg/kg, at least about 10 µg/kg, at least about 25 µg/kg, at least about 50 µg/kg, at least about 100 µg/kg, at least about 500 µg/kg, at least about 1 mg/kg , at least about 5 mg/kg, at least about 10 mg/kg, at least about 25 mg/kg, at least about 50 mg/kg, at least about 100 mg/kg, at least about 200 mg/kg, at least about 300 mg/kg, at least about 400 mg/kg, and at least about 500 mg/kg of the individual's body weight. In one embodiment, a dose is administered that provides a concentration of an anti-C5a antibody or antigen binding fragment thereof of the present invention of at least about 1 pM, at least about 10 pM, at least about 100 pM, at least about 1 nM , at least about 10 nM, at least about 100 nM, at least about 1 μM, at least about 2 μM, at least about 3 μM, at least about 4 μM, at least about 5 μM, at at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, and at least about 10 μM in an individual. In yet another embodiment, the invention provides for the administration of a dose that provides a concentration of an anti-C5a antibody or antigen binding fragment thereof of the present invention of at least about 1 pM, at least about 10 pM, at least about 100 pM, at least about 1 nM, at least about 10 nM, at least about 100 nM, at least about 1 μM, at least about 2 μM, at least about 3 μM, at least about 4 μM, at least about 5 μM , at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, and at least about 10 μM in the plasma of an individual.

In some embodiments, pharmaceutical compositions suitable for the practice of the invention may be administered to deliver a dose of no more than about 1 ng/kg, no more than about 5 ng/kg, no more than about 10 ng/kg, no more than about 25 ng/kg, not more than about 50 ng/kg, not more than about 100 ng/kg, not more than about 500 ng/kg, not more than about 1 μg/kg, not more than about 5 μg/kg , no more than approximately

- 17 043843 μg/kg, not more than about 25 μg/kg, not more than about 50 μg/kg, not more than about 100 μg/kg, not more than about 500 μg/kg, not more than about 1 mg/ kg, not more than about 5 mg/kg, not more than about 10 mg/kg, not more than about 25 mg/kg, not more than about 50 mg/kg, not more than about 100 mg/kg, not more than about 200 mg/kg, no more than about 300 mg/kg, no more than about 400 mg/kg, and no more than about 500 mg/kg of the individual's body weight. In one embodiment, a dose is administered that provides a concentration of the anti-C5 antibody of the present invention of no more than about 1 pM, no more than about 10 pM, no more than about 100 pM, no more than about 1 nM, no more than about 10 nM, not more than about 100 nM, not more than about 1 μM, not more than about 2 μM, not more than about 3 μM, not more than about 4 μM, not more than about 5 μM, not more than about 6 μM , not more than about 7 μM, not more than about 8 μM, not more than about 9 μM, and not more than about 10 μM, in an individual. In yet another embodiment, the invention provides for administration of a dose that provides a concentration of the anti-C5a antibody of the present invention of no more than about 1 pM, no more than about 10 pM, no more than about 100 pM, no more than about 1 nM, not not more than about 10 nM, not more than about 100 nM, not more than about 1 μM, not more than about 2 μM, not more than about 3 μM, not more than about 4 μM, not more than about 5 μM, not more less than about 6 μM, no more than about 7 μM, no more than about 8 μM, no more than about 9 μM, and no more than about 10 μM in the plasma of an individual. Dose ranges between any of the doses disclosed herein are also provided.

Typically, dosages that can be administered in the method of the invention to an individual, in some embodiments a human, range from 0.5 μg to about 50 mg per kg of the individual's body weight. Although the exact dosage administered will vary depending on a number of factors, including, but not limited to, the type of individual and the type of disease state being treated, the age of the individual and the route of administration. In some embodiments, the dosage of the compound will range from about 1 μg to about 10 mg per kg of body weight of the individual. In still other embodiments, the dosage will range from about 3 μg to about 1 mg per kg of body weight of the individual.

The antibody may be administered to the individual as often as several times a day, or it may be administered less frequently, such as once a day, twice a day, three times a day, once a week, twice a week, three times a week , once every two weeks, twice every two weeks, three times every two weeks, once a month, twice a month, three times a month or even less often, for example once every few months or even once or more times a a year or less. The frequency of dosing will be apparent to one skilled in the art and will depend on a number of factors such as, but not limited to, the type and severity of the disease being treated, the type and age of the individual, etc. The pharmaceutical compositions can be formulated by any method known or hereafter developed in the field of pharmacology. Typically, such preparation methods include the step of combining the active ingredient with a carrier or one or more other auxiliary ingredients, and then, if necessary or desired, forming or packaging the product into the desired single or multi-dose unit.

Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions that are suitable for ethical administration to humans, one skilled in the art will appreciate that such compositions are generally suitable for administration to individuals of all kinds. Modification of pharmaceutical compositions suitable for administration to humans to provide compositions suitable for administration to various individuals is well understood, and a veterinary pharmacologist of ordinary skill can develop and carry out such modification by simply routine, if any, experimentation. Subjects to whom the pharmaceutical compositions of the invention are contemplated include, but are not limited to, humans and other primates, mammals, including commercially important mammals such as non-human primates, cattle, pigs, horses, sheep, cats and dogs.

Pharmaceutical compositions that are useful in the methods of the invention can be formulated, packaged or marketed in formulations suitable for ophthalmic, oral, rectal, intravaginal, parenteral, topical, pulmonary, intranasal, buccal, intraocular, intravitreal, intramuscular, intradermal and intravenous route of administration. Other envisaged formulations include engineered nanoparticles, liposomal formulations, active ingredient loaded and released red blood cells, and immunological formulations.

The pharmaceutical composition of the invention may be formulated, packaged or marketed as a single dose or multiple unit doses. A unit dose represents a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient that is administered to the individual, or a convenient portion of such dose, such as one-half or one-third of such dose.

The relative amounts of the active ingredient, pharmaceutically acceptable carrier and any additional ingredients in the pharmaceutical composition of the invention will vary depending on the identity, size and condition of the individual being treated and, further, depending on the route by which the composition is to be administered . For example, the composition may contain from

0.1 to 100% (w/w) active ingredient. In various embodiments, the composition contains at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28 %, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35 %, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42% , at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, according to at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85 %, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92% , at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% (w/w) active ingredient.

In addition to the active ingredient, the pharmaceutical composition of the invention may further contain one or more additional pharmaceutically active agents.

It is possible to prepare controlled or sustained release formulations of the pharmaceutical composition of the invention using conventional technology.

Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical disruption of the integrity of the tissue of an individual and administration of the pharmaceutical composition through such disruption to the tissue.

Parenteral administration may be local, regional or systemic. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, administration of the composition through a surgical incision, administration of the composition through a tissue penetrating non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, intravenous, intraocular, intravitreolar, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, and intratumoral administration.

Formulations of a pharmaceutical composition suitable for parenteral administration include the active ingredient in combination with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline solution. Such formulations may be formulated, packaged or marketed in a form suitable for bolus administration or for continuous administration. Injectable formulations may be formulated, packaged or marketed in unit dosage form, such as ampoules, or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and sustained release or biodegradable implantable formulations. Such formulations may further contain one or more additional ingredients, including, but not limited to, suspending, stabilizing or dispersing agents. In one embodiment of a parenteral formulation, the active ingredient is provided in dry (ie, powder or granular) form for reconstitution with a suitable vehicle (eg, sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be formulated, packaged or marketed in the form of a sterile injectable aqueous or oily suspension or solution. Such a suspension or solution may be formulated in accordance with the prior art and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents or suspending agents. Such sterile injectable formulations can be prepared using a non-toxic parenterally acceptable diluent or solvent, such as, for example, water or 1,3-butanediol. Other suitable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fatty oils such as synthetic mono- or diglycerides. Other parenteral formulations that are suitable include those that contain the active ingredient in microcrystalline form, in a liposomal formulation, or as a component of biodegradable polymer systems. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as an emulsion, an ion exchange resin, a poorly soluble polymer, or a poorly soluble salt.

The pharmaceutical composition of the invention may be formulated, packaged or marketed in a formulation suitable for pulmonary administration through the oral cavity. Such a composition may contain dry particles that contain the active ingredient and that have a diameter ranging from about 0.5 nm to about 7 nm, and in some embodiments, from about 1 nm to about 6 nm. Such compositions are conveniently in the form of dry powders for administration using a device containing a dry powder reservoir into which a stream of propellant can be directed to disperse the powder, or using a self-propelled solvent/powder dispensing container, such as a device containing the active ingredient, dissolved or suspended in a low-boiling propellant in an airtight container. In some embodiments, such powders contain particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by weight have a diameter of less than 7 nm. In some embodiments, at least 95% of the particles by mass have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter of less than 6 nm. In some embodiments, the dry powder compositions include a solid, finely divided powder diluent, such as sugar, and are conveniently administered in unit dosage form.

Low boiling point propellants generally include liquid propellants having a boiling point at atmospheric pressure below 65°F. Typically, the propellant may comprise from 50 to 99.9% (w/w) of the composition, and the active ingredient may comprise from 0.1 to 20% (w/w) of the composition. The propellant may also contain additional ingredients, such as a liquid nonionic or solid anionic surfactant or a solid diluent (in some embodiments, having a particle size of the same order as the particles containing the active ingredient).

The pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of drops of a solution or suspension. Such formulations may be formulated, packaged or marketed as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, containing the active ingredient and may be conveniently administered using any spray or nebulization device. Such formulations may further contain one or more additional ingredients, including, but not limited to, a flavoring agent such as sodium saccharinate, a volatile oil, a buffering agent, a surfactant, or a preservative such as methyl hydroxybenzoate. In some embodiments, the droplets provided by injection have an average diameter ranging from about 0.1 nm to about 200 nm.

The compositions are also suitable for intranasal delivery of the pharmaceutical composition of the invention.

Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of from about 0.2 to 500 microns. Such a composition is administered in the same way as snuff is used, i.e. by quickly inhaling through the nasal passage from a container of powder held close to the nostrils.

Formulations suitable for intranasal administration may, for example, contain from about 0.1 to 100% (w/w) active ingredient and may also contain one or more additional ingredients.

The pharmaceutical composition of the invention may be formulated, packaged or marketed in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges prepared using conventional methods and may, for example, contain from 0.1 to 20% (w/w) of the active ingredient, the remainder being soluble or disintegrable in an oral composition and optionally one or more additional ingredients. Alternatively, formulations suitable for buccal administration may comprise a powder or an aerosolized or nebulized solution or suspension containing the active ingredient. In some embodiments, such powder, aerosolized, or spray formulations, when dispersed, have an average particle or droplet size ranging from about

0.1 nm to about 200 nm and may also contain one or more additional ingredients.

As used herein, additional ingredients include, but are not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricants; sweeteners; flavorings; dyes; preservatives; physiologically degradable compositions such as gelatin; aqueous carriers and solvents; oil carriers and solvents; suspending agents; dispersing or wetting agents; emulsifiers, softening agents; buffers; salt; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other additional ingredients that may be included in the pharmaceutical compositions of the invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein document by reference.

Cells that produce antibodies and their antigen-binding fragments.

In some embodiments, the invention is a cell or cell line (such as host cells) that produces at least one of the anti-C5a antibodies or antigen binding fragments described herein. In one embodiment, the cell or cell line is a genetically modified cell that produces at least one of the anti-C5a antibodies or antigen binding fragments described herein. In one embodiment, the cell or cell line is a hybridoma that produces at least one of the anti-C5a antibodies or antigen binding fragments described herein.

Hybrid cells (hybridomas) are typically derived from bulk fusions of mouse splenocytes, highly enriched in B cells, and myeloma cell fusion partners (Alberts et al., Molecular Biology of the Cell (Garland Publishing, Inc. 1994); Harlow et al., Garland Publishing, Inc. 1994); Harlow et al., Antibodies. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). The cells in the confluence are then sorted into pools that can be analyzed for antibody production with the desired specificity. Pools that test positive can be further subdivided until single cell clones are identified that produce antibodies with the desired specificity. Antibodies produced by such clones are called monoclonal antibodies.

Also provided are nucleic acids encoding any of the antibodies or antibody fragments disclosed herein, as well as vectors containing the nucleic acids. Thus, antibodies and fragments of the invention can be produced by expressing a nucleic acid in a cell or cell line, such as cell lines typically used for the expression of recombinant or humanized immunoglobulins. Thus, antibodies and fragments of the invention can also be prepared by cloning nucleic acids into one or more expression vectors and transforming the vector into a cell line, such as cell lines typically used for the expression of recombinant or humanized immunoglobulins.

Genes encoding immunoglobulin heavy and light chains or fragments thereof can be engineered according to methods including, but not limited to, polymerase chain reaction (PCR) known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y., 1989; Berger & Kimmel, Methods in Enzymology, vol. 152: Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego, Calif., 1987; Co et al., 1992, J. Immunol., 148:1149). For example, genes encoding heavy and light chains or fragments thereof can be cloned from the genomic DNA of an antibody-secreting cell, or the cDNA can be obtained by reverse transcription of cellular RNA. Cloning is carried out by conventional methods, including the use of PCR primers that hybridize to sequences flanking or overlapping the genes or gene segments to be cloned.

Nucleic acids encoding an antibody of the invention, or a heavy chain or light chain or fragments thereof, can be prepared and used in accordance with recombinant nucleic acid techniques to produce a particular immunoglobulin, immunoglobulin chain, or fragment or variant thereof, in various host cells or in an in vitro translation system. For example, antibody-encoding nucleic acids, or fragments thereof, can be placed into suitable prokaryotic or eukaryotic vectors, such as expression vectors, and introduced into a suitable host cell by an appropriate method, for example, transformation, transfection, electroporation, infection such that the nucleic acid becomes operably linked to one or more expression regulatory elements, for example, in a vector or integrated into the genome of a host cell.

In some embodiments, the heavy and light chains or fragments thereof can be placed into two different expression vectors that can be used to co-transfect a recipient cell. In some embodiments, each vector may contain two or more selectable genes, one for selection in a bacterial system and one for selection in a eukaryotic system. Such vectors enable gene production and amplification in a bacterial system, as well as subsequent cotransfection of eukaryotic cells and selection of cotransfected cells. A selection procedure can be used to select the expression of antibody nucleic acids introduced in two different DNA vectors into a eukaryotic cell.

Alternatively, nucleic acids encoding the heavy and light chains or fragments thereof can be expressed from a single vector. Although the light and heavy chains are encoded by separate genes, they can be combined using recombinant techniques. For example, two polypeptides can be joined by a synthetic linker that allows them to form a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al., 1988 , Science, 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci., USA, 85:5879-5883.

The invention provides an isolated nucleic acid molecule containing a nucleic acid sequence encoding a heavy chain and/or a light chain, as well as fragments thereof. A nucleic acid molecule containing sequences encoding both the light and heavy chain, or fragments thereof, can be designed to contain a synthetic signal sequence for secretion of the antibody or fragment when produced in a cell. In addition, the nucleic acid molecule may contain specific DNA linkages that allow the insertion of other antibody sequences and maintain the translational reading frame so as not to alter amino acids typically found in antibody sequences.

In accordance with the present invention, nucleic acid sequences encoding an antibody can be inserted into a suitable expression vector. In various embodiments, the expression vector contains the necessary elements for transcription and translation of the inserted antibody-encoding nucleic acid so as to generate recombinant DNA molecules that direct expression of the antibody sequences to produce an antibody or fragment thereof.

Nucleic acids encoding antibodies or fragments thereof can be subjected to various recombinant nucleic acid techniques known to those skilled in the art, such as site-directed mutagenesis.

A variety of methods can be used to express nucleic acids in a cell. Nucleic acids can be cloned into a number of different types of vectors. However, the present invention should not be construed as limited to any particular vector. Instead, the present invention should be considered to cover a wide range of vectors that are readily available and/or known in the art. For example, a nucleic acid of the invention can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector, and a mammalian cell vector. There are numerous vector expression systems that include at least some or all of the compositions discussed above. Prokaryotic and/or eukaryotic vector systems can be used for use in the present invention to produce polynucleotides or related polypeptides thereof. Many such systems are commercially and widely available.

Viral vector technology is well known in the art and is described, for example, in the monographs of Sambrook et al. (2012) and Ausubel et al. (1999) and other textbooks on virology and molecular biology. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses and lentiviruses. In some embodiments, a murine stem cell virus (MSCV) vector is used to express the desired nucleic acid. MSCV vectors have been shown to efficiently express desired nucleic acids in cells. However, the invention should not be limited to the use of the MSCV vector only, but rather any retroviral expression method is included in the invention. Other examples of viral vectors are those based on Moloney murine leukemia virus (MoMuLV) and HIV. In some embodiments, a suitable vector comprises an origin of replication functional in at least one organism, a promoter sequence, convenient restriction sites, and one or more selectable markers (see, for example, WO 01/96584; WO 01/29058; and US Pat. No. 6326193).

Additional regulatory elements can be used, such as enhancers that modulate the frequency of transcription initiation. A promoter can be an element that is naturally associated with a gene or nucleic acid sequence, which can be obtained by isolating 5' non-coding sequences located upstream of the coding segment and/or exon.

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Such a promoter may be an endogenous promoter. Likewise, an enhancer may be an enhancer that is naturally associated with a nucleic acid sequence located downstream or upstream of that sequence. Alternatively, certain advantages are obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not typically associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cells, and promoters or enhancers not found in nature, for example, containing different elements of different transcriptional regulatory regions, and /or mutations that change expression. In addition to obtaining promoter and enhancer nucleic acid sequences synthetically, sequences can be obtained using recombinant cloning and/or nucleic acid amplification technology, including PCR, for the compositions disclosed herein (U.S. Patent No. 4,683,202; U.S. Patent No. 5,928,906). In addition, it is believed that control sequences can also be used to directly transcribe and/or express sequences within nonnuclear organelles such as mitochondria, chloroplasts, and the like.

Of course, it is important to use a promoter and/or enhancer that effectively directs expression of the DNA segment in the cell type, organelle and organism selected for expression. Molecular biologists typically know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012). The promoters used may be constitutive, tissue specific, inducible and/or suitable under appropriate conditions for directing high-level expression of the introduced DNA segment, which is advantageous in large-scale production of recombinant proteins and their fragments.

An example of a promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, viral promoter Moloney, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter and muscle creatine promoter. Moreover, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also provided as part of the invention. The use of an inducible promoter in the invention provides a molecular switch capable of turning on expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. The invention further includes the use of a tissue-specific promoter or a cell type-specific promoter, which is a promoter that is active only in the desired tissue or cell. Tissue-specific promoters are well known in the art and include, but are not limited to, the HER-2 promoter and PSA-linked promoter sequences.

To assess nucleic acid expression, the expression vector to be introduced into a cell may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the population of cells that are being transfected or infected with viral agents. vectors. In other embodiments, the selectable marker may be carried in a separate nucleic acid and used in a cotransfection procedure. Both selectable marker genes and reporter genes can be flanked by appropriate regulatory sequences to allow expression in host cells. Suitable selectable marker genes are known in the art and include, for example, antibiotic resistance genes such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Reporter genes that encode proteins that are easily analyzed are well known in the art. In general, a reporter gene is a gene that is absent or not expressed by the recipient organism or tissue and that encodes a protein whose expression results in some easily detectable property, such as enzymatic activity. Expression of the reporter gene is analyzed at the appropriate time after introduction of DNA into recipient cells.

- 23 043843

Suitable reporter genes may include genes encoding luciferase, betagalactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein gene (see, for example, Ui-Tei et al., 2000, FEBS Lett., 479:79-82). Suitable expression systems are well known and can be prepared using well known techniques or obtained from commercial sources. In general, the construct with the minimal 5' flanking region showing the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

Methods for introducing and expressing nucleic acids into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, such as a mammalian, bacterial, yeast, or insect cell, by any method known in the art. For example, the expression vector can be introduced into a host cell by physical, chemical or biological means.

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, laser poration, and the like. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012), Ausubel et al. (1999).

Biological methods for introducing a nucleic acid of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, US Pat. No. 5,350,674 and US Pat. No. 5,585,362.

Chemical means for introducing nucleic acid into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle). The preparation and use of such systems are well known in the art.

Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to a nucleic acid of the present invention, various assays can be performed to confirm the presence of a recombinant DNA sequence in a host cell. Such assays include, for example, molecular, biological assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a specific peptide, for example, immunological methods (ELISA and Western blotting) or assays described herein, to identify agents within the scope of the invention.

Sets.

The invention also includes a kit containing an anti-C5a antibody or combinations thereof of the invention and instructional material that describes, for example, administering an anti-C5a antibody or combinations thereof to an individual as a therapeutic treatment or use other than treatment, as described elsewhere in this document. In one embodiment, the kit further comprises an (optionally sterile) pharmaceutically acceptable carrier suitable for dissolving or suspending a therapeutic composition comprising the anti-C5a antibody or combinations thereof of the invention, for example, prior to administration of the antibody to a subject. Optionally, the kit includes an applicator for administering the antibody.

Experimental examples.

The invention will now be described with reference to the following examples. These examples are provided for the purpose of illustration only, and the invention should in no way be construed as being limited by these examples, but rather should be construed as covering any and all variations that become apparent from reading the inventive concept presented herein.

Without further description, it is believed that one skilled in the art, using the foregoing description and the following illustrative examples, can prepare and use the compounds of the present invention and practice the claimed methods. Accordingly, the following worked examples should not be construed as limiting in any way the remainder of the disclosure.

Example.

The complement system is part of the innate immune system, which plays a key role in host defense. However, activated complement also has the potential to cause significant tissue damage and destruction, and dysregulation of complement activity has been found to be associated with a number of rare and common diseases such as paro

- 24 043843 xysmal nocturnal hemoglobinuria (PNH), atypical hemolytic-uremic syndrome, rheumatoid arthritis, age-related macular degeneration, etc. Thus, anticomplement therapy is a promising treatment option for these human disorders. The C5 component of the complement system is a critical protein in the terminal pathway of complement activation and is a precursor protein in the generation of the potent proinflammatory mediator C5a as well as the cytolytic membrane attack complex (MAC), C5b-9.

A function-blocking, anti-human C5a monoclonal antibody (7A12) was developed and described herein. This mAb blocks C5a-mediated activity but does not block MAC activity (as determined by hemolytic assay).

The methods and materials used in this example will now be described.

Western blotting.

Purified human C5 protein (1 μg) was boiled in sample buffer and loaded onto 6% SDS-PAGE gels under nonreducing or reducing conditions. Proteins were transferred to PVDF membranes, which were probed with 10 μg/ml primary antibody (mAb 7A12 or control anti-C5 mAb) for 1 h, followed by detection using HRP-conjugated anti-mouse IgG (1:4000, Bio-Rad).

Human C5, C5a and mAb binding assay.

Polystyrene microtiter plates were coated with purified human C5 or C5a (50 ng/well, Hycult) in PBS at 37°C for 1 h. After aspiration of the C5 or C5a solution, the wells were blocked with PBS containing 1% BSA in PBS at room temperature for 1 hour. Wells without C5 or C5a coating served as background controls. Various concentrations of mAb 7A12, or control anti-C5 mAb, or chimeric 7A12, were added to the wells at 50 μl/well in blocking solution. After incubation for 1 h at room temperature, the wells were washed thoroughly with PBST. Human C5 or C5a bound mAbs were detected by adding HRP-conjugated anti-mouse IgG at a dilution of 1:4000 in blocking solution, which was allowed to incubate for 1 hour at room temperature. After washing with PBST, the plate was developed with HRP substrate for 6-10 min. The reaction was stopped by adding 2 N H ₂ SO ₄ solution and the plate was analyzed at 450 nm on a microplate reader.

Mouse C5a and mAb binding assay.

Polystyrene microtiter plates were coated with purified mouse C5a (50 ng/well, Hycult) in PBS at 37°C for 1 h. After aspiration of the C5a solution, the wells were blocked with PBS containing 1% BSA in PBS at room temperature for 1 h. uncoated C5a served as background control. Various concentrations of mAb 7A12 or control anti-C5a mAb or chimeric 7A12 were added to the wells at 50 μl/well in blocking solution. After incubation for 1 h at room temperature, the wells were washed thoroughly with PBST. Murine C5a-bound mAbs were detected by adding HRP-conjugated anti-mouse IgG at a 1:4000 dilution in blocking solution, which was allowed to incubate for 1 h at room temperature. After washing with PBST, the plate was developed with HRP substrate for 6-10 min. The reaction was stopped by adding 2 N H2SO4 solution and the plate was analyzed at 450 nm on a microplate reader.

Preparation of antibodies against human C5a.

Female B10.D2/oSnJ mice (Stock #000461, Jackson Nursery) were immunized with 30 μg of purified human C5 (#A120, Complement Technology, Inc) emulsified with adjuvant. On day 14, mice were again immunized with 30 μg of purified human C5 emulsified with adjuvant. Mice were injected with 33 μg of purified human C5 three times before fusion. Mice were then sacrificed by cervical dislocation and the spleen was isolated to prepare a single cell suspension by mechanical disruption. The spleen cell suspension was washed once with serum-free hybridoma medium (HYB-SFM) (Invitrogen) + 10% FBS medium and the cells were counted and mixed with X63-Ag8.653 myeloma cell line (ATCC) in a 2:1 ratio. The cell mixture was again washed with HYB-SFM medium and the cell pellet was obtained by centrifugation (1000 rpm, x5 min). The cell pellet was gently disturbed and loosened, and cell fusion was then induced by slowly adding polyethylene glycol (PEG 1500) (1.5 ml PEG per 3 x 10 ⁸ cells). The cells were left for 1 minute at 37°C and then 20 ml of HYB-SFM medium was added to the cells over 3 minutes (1 ml in the first minute, 3 ml in the second minute and 16 ml in the third minute). The mixture was centrifuged at 1000 rpm for 5 min and cells were seeded in 24-well plates in HAT medium (10 ml HAT [Sigma H0262], 5 ml Pen/Strep, 500 μl gentamicin and 10% FBS in 500 ml HYB- SFM). After 2 weeks, supernatants from wells with visible colonies were collected for reactivity screening with purified human C5 by ELISA. Positive clones were collected and seeded into 96-well plates by serial dilution to obtain individual clones after a second round of ELISA screening. Positive clones were propagated in HT medium (10 ml HT, 5 ml Pen/Strep, 500 μl gentamicin and 10% FBS in 500 ml HYB-SFM medium). Before collecting antibodies, hybridoma cells were transferred to serum-free medium (SF-SFM) for 2-3 days. Cell culture medium was collected for mAb purification using

- 25 043843 affinity chromatography with protein G.

mAb cloning.

To clone 7A12 cDNA, the total RNA fraction was isolated from hybridoma cells using the TRIzol reagent (Sigma). First-strand cDNA was synthesized by reverse transcription using the Oligo(dT) primer. To amplify heavy chain cDNA (for IgG1, IgG2a/b) in PCR reactions, the following primers were used: 5'-GAG GTG A AGCTG GTG G AG (T/A) C (T/A) GG-3' (SEQ ID NO: 11) and 5'-GGGGCCAGTGGATAGAC-3' (SEQ ID NO: 12). The following primers were used for light chain amplification:

mixture of 4 direct primers:

5'-CCAGTTCCGAGCCAGATGATGACCCAGACTCCA-3 '(SEQ ID NO: 70), 5'-CCAGTTCCGAGCTCCCACCCAGTCCCA-3' (Seq ID NO: 71), 5'-CCAGTTCCCAGACCCCCCCCC AGTCTCCA-3 '(SEQ ID NO: 72), 5'-CCAGTTCCGAGTGATGATGTCCCCA- 3' (SEQ ID NO: 13); reverse primer: 5'-GTTGGTGCAGCATCAGC-3' (SEQ ID NO: 14).

PCR amplicons were cloned into the pCR TOPO TA 2.1 vector (Invitrogen) and sequenced. To obtain the signal peptide (leader) sequence of the mAb, the 5'-RACE method with a kit (GeneRacer) from Invitrogen was used. Full variable region cDNAs were amplified using specific primers determined using the 5′-RACE method and the raw sequencing data.

Hemolysis assay.

Antibody-sensitized sheep red blood cells (1 x 10 ⁷ cells, Complement Technology, Inc.) were incubated at 37°C for 20 min with 50% NHS (Complement Technology, Inc) in gelatin veronal buffer (GVB2+, Sigma). Before addition to sheep red blood cells, NHS was preincubated with 7A12 mAb or control anti-C5 mAb for 1 h at 4°C. The lysis reaction was stopped by adding an ice-cold 40 mM EDTA solution in PBS. Incubation mixtures were centrifuged for 5 min at 1500 rpm, the supernatant was collected and measured at OD405 nm. Samples without added NHS or with EDTA were used as negative lysis controls and a sample of sheep red blood cells completely lysed with distilled water was used as a positive control (100% lysis) against which the percentage of lysis in the other samples was normalized.

Migration analysis in the Transwell system.

U937 cells stably transfected with the C5a receptor (U937-C5aR cells) were seeded (at 1 × 10 ⁶ cells per well in chemotaxis assay buffer) into the upper chambers of 24-well Transwell inserts with a 3.0 μm polycarbonate membrane filter (Corning ). The lower Boyden chambers were filled with 10 nM recombinant human complement C5a pretreated with antibody (1 or 10 μg/ml) in chemotaxis assay buffer. After incubation for 3 h at 37°C, migrated cells in the lower chambers were collected and counted using a Beckman Coulter counter. (Chemotaxis assay buffer: RPMI 1640 medium with 0.5% BSA.)

Calcium mobilization assay.

1x10 ⁷ cells were washed twice with HEPES calcium mobilization assay buffer (25 mM HEPES, 119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl ₂ and 1 mM CaCl ₂ ) containing 1 mg/ml BSA, and then incubated with 1 μM indo-1-acetoxymethyl ether (Anaspec, Inc, Fremont, CA, USA) at room temperature for 30 min. The cells were then washed twice and resuspended in 1.3 ml of the same buffer. Human C5a and antibody were mixed to a final concentration of 10 nM and 50 μg/ml, respectively, for 10 min at room temperature. Finally, intercellular Ca ²⁺ measurements were performed using an Infinite F200 multimode microplate reader (Tecan Systems Inc, San Jose, CA, USA) with an excitation wavelength of 360 nm and an emission wavelength of 415 nm. The mixture was added 300 s after the start of the measurement.

The results of this example are now described.

As shown in FIG. 1,7A12 has been shown to bind to human C5. The reactivity of 7A12 and 2G1, a control anti-C5 mAb, with intact human C5 was assessed by ELISA as shown in FIG. 1. The ELISA plate was coated with purified human C5. After incubation with serial dilutions of 7A12 or control anti-C5 mAb, bound mAb was detected using HRP-conjugated rabbit anti-mouse IgG. 7A12 and control anti-C5a mAb showed high reactivity with human C5. Using Western blotting, mAb 7A12 and control anti-C5a mAb were found to recognize purified human C5 protein under non-reducing and reducing conditions, respectively, as shown in FIG. 1B. The observed 7A12-reactive band of 190 kDa represents the complete C5 protein, whereas the 115 kDa band represents the C5 α chain.

In contrast to the control anti-C5 mAb 2G1, which inhibited red blood cell (RBC) lysis, 7A12 was shown to be inactive in the hemolysis assay as shown in FIG. 2. Antibody-sensitized sheep red blood cells were incubated with normal human serum

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Claims (9)

(NHS) containing serial dilutions of 7A12 or control anti-C5 mAb at 37°C for 1 hour. RBC lysis was determined by measuring optical density at OD405 nm. As expected, the control anti-C5 mAb inhibited NHS-mediated lysis of ovine erythrocytes by 50% in a dose-dependent manner. On the other hand, at doses ranging from 0.975 to 120 μg/ml, mAb 7A12 did not show 50% inhibition of NHS-mediated lysis of ovine erythrocytes. In contrast to the control anti-C5 mAb 2G1, which does not bind to C5a, 7A12 was shown to bind to human C5a. The ELISA plate was coated with human C5a or mouse C5a. After incubation with serial dilutions of 7A12 or control anti-C5 mAb, bound mAb was detected using HRP-conjugated anti-mouse IgG. In fig. 3A shows that mAb 7A12 was highly reactive to human C5a. Since mAb 7A12 reacts with the complete C5 protein (as shown in Fig. 1), it can be concluded that mAb 7A12 binds to both the C5a fragment of native human C5 and free human C5a. On the other hand, control anti-C5 mAb was not found to bind to human C5a as shown in FIG. 3B. Binding of mAb 7A12 to C5a was found to be specific to human C5a, with little binding to mouse C5a. Binding affinity of mAb 7A12 to human C5 and C5a. Purified human C5 or C5a was coupled to the CM4 chip using the amine coupling method. Biacore analysis was performed on a Biacore-2000 instrument. Between each binding, the chip was regenerated using 50 mM NaOH. mAb 7A12 binds to human C5 as shown in FIG. 4A, and human C5a, as shown in FIG. 4B, with similar affinity. The results shown in FIG. 5 demonstrate that mAb 7A12, but not the control anti-C5 mAb 2G1, inhibits C5a-mediated neutrophil migration. Human C5a at a concentration of 10 nM was used to induce chemotaxis of a human monocytic cell line, U937, transfected with the human C5a receptor. Cells were placed in the upper chamber of the Transwell system in the presence of mAb 7A12 or control anti-C5 mAb 2G1, and cell migration was quantified by counting cells in the lower chamber. mAb 7A12 showed complete inhibition of C5a-induced chemotaxis at a concentration of 10 μg/ml, whereas the control anti-C5 mAb 2G1 failed to block C5a-induced chemotaxis. The results shown in FIG. 6 demonstrate that mAb 7A12, but not the control anti-C5 mAb 2G1, inhibits C5a-induced intracellular calcium mobilization in U937 cells. Calcium mobilization was absent in U937 cells expressing the human C5a receptor (U937-C5aR) in the absence of human C5a stimulation, as shown in FIG. 6A. Treatment with C5a resulted in a transient calcium influx in U937-C5aR cells, as shown in Fig. 6B, which could be inhibited by preincubation with mAb 7A12 (50 μg/ml) as shown in FIG. 6C, but not with the control anti-C5 mAb 2G1 (50 μg/ml) as shown in FIG. 6D. The arrows refer to the time point when the C5a and antibody mixture was added to the cell suspension. The disclosure of each patent, patent application and publication cited herein is incorporated by reference in its entirety. Although the present invention has been disclosed with reference to specific embodiments, it will be appreciated that other embodiments and variations of the present invention may be developed by others skilled in the art without departing from the spirit and scope of the invention. The appended claims are intended to include all such embodiments and equivalent embodiments. CLAIM 1 amino acid substitution; and iii) VL-CDR3 containing the amino acid sequence of SEQ ID NO: 10, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitution. 1 amino acid substitution; ii) VL-CDR2 containing the amino acid sequence of SEQ ID NO: 9, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or 1. An antibody that specifically binds to human C5a, wherein the antibody contains a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH contains i) VH-CDR1 containing the amino acid sequence of SEQ ID NO: 3, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitution; ii) VH-CDR2 containing the amino acid sequence of SEQ ID NO: 4, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitution; and iii) VH-CDR3 containing the amino acid sequence of SEQ ID NO: 5, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitution; and where VL contains i) VL-CDR1 containing the amino acid sequence of SEQ ID NO: 8, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or - 27 043843 2. An antibody according to claim 1, wherein the antibody is a chimeric antibody. 3. Antibody according to claim 1, where the antibody contains a CDR: V H-CDR1: SEQ ID NO: 3; V H-CDR2: SEQ ID NO: 4; V H-CDR3: SEQ ID NO: 5; V L-CDR1: SEQ ID NO: 8; VL-CDR2: SEQ ID NO: 9; And V L-CDR3: SEQ ID NO: 10. 4. The antibody of claim 1, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 2, or a variant thereof. 5. The antibody of claim 1, wherein the antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 7, or a variant thereof. 6. The antibody of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof, and a light chain containing the amino acid sequence of SEQ ID NO: 7, or a variant thereof. 7. A method of treating a complement-mediated disease or disorder in an individual, comprising the step of administering to said individual an anti-C5a antibody according to any one of claims 1 to 6. 8. The method of claim 7, wherein the disease or disorder is selected from the group consisting of macular degeneration (MD), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, postoperative syndrome systemic inflammatory response, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria syndrome (PNH), autoimmune hemolytic anemia (AIHA), Gaucher disease, myasthenia gravis, neuromyelitis optica, NMO, multiple sclerosis, delayed graft function , antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS), central retinal vein occlusion (CRVO), central retinal artery occlusion (CRAO), epidermolysis bullosa, sepsis, septic shock, organ transplantation, inflammation (including but not limited to inflammation associated with cardiopulmonary bypass and renal dialysis), C3 glomerulopathy, membranous nephropathy, IgA nephropathy, glomerulonephritis (including, but not limited to, antineutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis, lupus nephritis and combinations thereof), ANCA-associated vasculitis , Shiga toxin-induced HUS, and antiphospholipid antibody-induced pregnancy loss, graft-versus-host disease (GVHD), or any combination thereof. 9. A method of reducing the activity of an individual's complement system, wherein the method includes administering an antibody to an individual by an administration route selected from the group consisting of enteral administration, parenteral administration, and a combination thereof, and wherein the antibody contains VH and VL, wherein VH contains i) VH-CDR1 containing the amino acid sequence of SEQ ID NO: 3, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitution; ii) VH-CDR2 containing the amino acid sequence of SEQ ID NO: 4, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitution; and iii) VH-CDR3 containing the amino acid sequence of SEQ ID NO: 5, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitution; and where VL contains i) VL-CDR1 containing the amino acid sequence of SEQ ID NO: 8, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitution; ii) VL-CDR2 containing the amino acid sequence of SEQ ID NO: 9, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3, 2 or 1 amino acid substitution; and iii) VL-CDR3 containing the amino acid sequence of SEQ ID NO: 10, or a variant thereof, where the variant retains biological properties and contains conservative substitutions containing up to 3.2 -